We have shown that normal human gingival keratinocyte (NHGK) cells undergo terminal differentiation upon reaching confluency by high Ca++ in a similar fashion as in vivo. Induction of TGase 1 activity (5 to 10-fold increase) was followed by formation of insoluble cell envelopes (CEs) suggesting that TGase 1 is the key marker for terminal differentiation of oral keratinocytes. The mRNA levels of other markers of terminal differentiation, e.g. involucrin, SPR1 and annexin 1, were also increased in the terminally differentiating NHGK cells. SPR1, annexin 1, loricrin, envoplakin, desmoplakin, involucrin, cystatin alpha and pancornulin were identified as components of NHGK CEs. These CEs contained an unusually high amount of SPR1, suggesting an important role of SPR1 in the specialized barrier function of oral epithelium. We have compared the expression of cytokeratins 1, 5, 8, 10, 14 and 19, TGase I and TGase II in NHGK cells and several head and neck cancer cells (HN4, HN12, HN8, HN22, HN30, HN31, HN13 and HN19). The expression of the major cytokeratins (1, 5, 10 and 14) was significantly reduced in most HNSCC lines. Aberrant expression of cytokeratin 8 or 19 was observed in some carcinoma cells. The level of TGase 1 activity was very low (~10% of NHGK) in all oral SCC cell lines. In contrast to NHGK cells, HNSCC cells failed to induce TGase 1 and failed to form cornified cell envelopes, consistent with their lost ability for terminal differentiation. TGase 2 was not inducible but an unusually high level of TGase 2 was observed in HN12, HN30 and HN31 cells. The TGase 2 activity of these HN cells appeared to correlate with their ability to grow in soft agar, but not with the tumorigenicity in nude mice. We have immortalized NHEK cells using HPV16 E6/E7 genes. The immortalized human gingival keratinocytes (IHGK) show a similar pattern of cytokeratin expression, as do NHGK. Like NHGK cells, IHGK cells undergo terminal differentiation upon reaching post-confluency in a high Ca++ medium. However, the capacity of IHGK to induce TGase 1 and to form CEs seemed to decrease with the increased number of passages in culture. IHGK cells were transfected with various human proto-oncogenes, including H-Ras, N-Ras, K-Ras, c-Raf, and c-Myc, and the activated oncogenes, H-ras-V12, H-ras-D12, K-ras-V12, using the pCEFL vector, and G418-resistant cells were isolated. We are characterizing the growth properties of these oncogene-transfected cells in liquid and in soft agar, their Ca++ resistance, and their ability to form tumors in nude mice.